Immunohistochemistry (IHC) refers to the processes of detecting, localizing, and/or quantifying antigens, such as a protein, in a biological sample using specific binding moieties, such as antibodies specific to the particular antigens. IHC provides the substantial advantage of identifying exactly where a particular protein is located within the tissue sample. It is also an effective way to examine the tissues themselves. In situ hybridization (ISH) refers to the process of detecting, localizing, and quantifying nucleic acids. Both IHC and ISH can be performed on various biological samples, such as tissue (e.g. fresh frozen, formalin fixed, paraffin embedded) and cytological samples. Recognition of the targets can be detected using various labels (e.g., chromogenic, fluorescent, luminescent, radiometric), irrespective of whether the target is a nucleic acid or an antigen. To robustly detect, locate, and quantify targets in a clinical setting, amplification of the recognition event is desirable as the ability to confidently detect cellular markers of low abundance becomes increasingly important for diagnostic purposes. For example, depositing at the marker's sites hundreds or thousands of label molecules in response to a single antigen detection event enhances, through amplification, the ability to detect that recognition event.
Adverse events often accompany amplification, such as non-specific signals that are apparent as an increased background signal. An increased background signal interferes with the clinical analysis by obscuring faint signals that may be associated with low, but clinically significant, expressions. Accordingly, while amplification of recognition events is desirable, amplification methods that do not the increase background signal are highly desirable. One such method is Tyramide Signal Amplification (TSA), which has also been referred to as catalyzed reporter deposition (CARD). U.S. Pat. No. 5,583,001 discloses a method for detecting and/or quantitating an analyte using an analyte-dependent enzyme activation system that relies on catalyzed reporter deposition to amplify the detectable label signal. Catalysis of an enzyme in a CARD or TSA method is enhanced by reacting a labeled phenol molecule with an enzyme. Modern methods utilizing TSA effectively increase the signals obtained from IHC and ISH assays while not producing significant background signal amplification (see, for example, U.S. application publication No. 2012/0171668 which is hereby incorporated by reference in its entirety for disclosure related to tyramide amplification reagents). Reagents for these amplification approaches are being applied to clinically important targets to provide robust diagnostic capabilities previously unattainable (OPTIVIEW® Amplification Kit, Ventana Medical Systems, Tucson Ariz., Catalog No. 760-099).
TSA takes advantage of the reaction between horseradish peroxidase (HRP) and tyramide. In the presence of H2O2, tyramide is converted to a highly-reactive and short-lived radical intermediate that reacts preferentially with electron-rich amino acid residues on proteins. Covalently-bound detectable labels can then be detected by variety of chromogenic visualization techniques and/or by fluorescence microscopy. In solid-phase immunoassays such as IHC and ISH, where spatial and morphological context is highly valued, the short lifetime of the radical intermediate results in covalent binding of the tyramide to proteins on tissue in close proximity to the site of generation, giving discrete and specific signal. While CARD broadly defines the use of an analyte-dependent reporter enzyme (ADRE) to catalyze covalent binding of numerous detectable labels to proteins, HRP-based TSA is a commercially validated approach. No alternative ADRE systems exist despite a strong need in the field for alternative amplification systems.
U.S. Pat. No. 7,291,474 to Bobrow postulates using hydrolase-based CARD. In particular, Bobrow hypothesizes that the activity probes 2-difluoromethylphenyl and p-hydroxymandelic acid could be used as amplification reagents. The use of 2-difluoromethylphenyl and p-hydroxymandelic acid was described by Zhu et al., (2003) Tetrahedron Letters, 44, 2669-2672; Lo et al., (2002) J. Proteome Res., 1, 35-40; Cesaro-Tadic et al., (2003) Nature Biotechnology, 21, 679-685; Janda et al., (1997) Science 275, 945-948; Halazy et al., (1990) Bioorganic Chemistry 18, 330-344; and Betley et al., (2002) Angew. Chem. Int. Ed. 41, 775-777. Bobrow's suggested structures included the following:
wherein Y is a moiety capable of being cleaved by a hydrolytic enzyme; L is a detectable label; X is a linking group; Z is a halogen; and R is hydrogen, alkyl, or halogen. In specific embodiments, R is hydrogen and the Z groups are fluorine. These structures are generalizations of the particular structures disclosed by Zhu et al., (2003) Tetrahedron Letters, 44, 2669-2672. In particular, Zhu et al. describes the following structures as known phosphatase inhibitors:

Based on these phosphatase inhibitors, Zhu et al. developed the following activity probes:
Zhu discloses that the activity-based profiling of proteins is a proven and powerful tool in proteomic studies, whereby subclasses of enzymatic proteins can be selectively identified. As such, Zhu developed the activity probes to signal the presence of active phosphatase enzymes. Zhu's strategy takes advantage of specific probes that react with different classes of enzymes, leading to the formation of covalent probe-protein complexes that are readily distinguished from other non-reactive proteins in a crude proteome mixture.
Zhu et al. state that it was known that 2-difluoromethylphenyl phosphate was a general phosphatase inhibitor against a broad spectrum of different phosphatases, including acid and alkaline phosphatases. Inhibition occurs as the phosphatases catalyze phosphate group cleavage to generate a reactive intermediary after a fluoride ion leaves. The reactive intermediary reacts with the enzyme's active site to covalently bind a fluorophore to the enzyme active site. But in doing so, it also inhibits the enzyme's ability to further hydrolytically cleave phosphates.
Using enzyme inhibitors in an amplification scheme to covalently bind signal generating moieties to a substrate is understood to be self-limiting as the generation of bound signal can destroy the activity of the enzyme. There has been a recognition in the art that pursuing enhancements made to these reagents would likely be self-defeating as the improved performance (e.g. turnover, specificity) would result in more efficient destruction of the enzyme's active site. Thus, in order to get signal amplification by binding multiple signal generating moieties, multiple enzymes first have to be bound proximally to the target. Accordingly, the compounds disclosed by Zhu et al. and Bobrow have never been developed into a commercially viable detection reagent for an amplification system for IHC or ISH.
Furthermore, the amplification approaches described thus far enable the deposition of fluorescent compounds. Fluorescence imaging is often implemented because it is extraordinarily sensitive; the detection of very few fluorophore molecules is now routine. However, this sensitivity is achieved using dark-field imaging, which has certain pragmatic limitations. For example, bright-field primary staining (e.g., hematoxylin and eosin staining) cannot be concurrently observed, making it more difficult to correlate fluorescent signal with morphological features. It is well known that fluorescence-based detection is routinely 1000 times more sensitive than absorbance-reflectance-based approaches (e.g. chromogenic-based detection). As such, a methodology appropriate for fluorescence detection would require a 1000-fold improvement for use as a chromogenic detection methodology. Increasing the performance of an enzyme-based detection system by 1000-fold is non-trivial. To date, only tyramide-based systems have achieved this increased performance.
While robust reagents are available, a need persists for alternative signal amplification approaches that produce robust amplification without increasing background signals. Moreover, methods for amplifying the detection of two or more distinct targets in a tissue sample are desirable.